Académique Documents
Professionnel Documents
Culture Documents
Springer-Verlag 1997
ORIGINAL PAPER
Received: 4 November 1996 / Received revision: 24 February 1997 / Accepted: 28 February 1997
Introduction
The revolution in molecular biology during the last decades has provided novel tools for studies of microbial
ecology. The availability of culture-independent methods like polymerase-chain-reaction-assisted direct cloning (PCR cloning), sequencing of rRNA genes, and in
situ hybridization has changed our view about the diversity and species composition of natural microbial
communities (Giovannoni et al. 1990; Amann et al.
1991; Fuhrman et al. 1992; DeLong et al. 1989; Olsen
et al. 1994).
The bacterial communities inhabiting activated
sludge plants have been studied for almost a century by
analyzing culturable bacterial isolates (Russel and
Bartow 1916; Buttereld 1935; Allen 1944; Dias and
Bhat 1964; Benedict and Carlson 1971; Blaim et al. 1984;
Hantula et al. 1991a). As a result, they are known to be
very diverse and dynamic systems, where proteobacteria
predominate. A novel insight to this community was
obtained when studies based on PCR cloning and in situ
hybridization were carried out (Wagner et al. 1993;
Manz et al. 1994; Wagner et al. 1994a, b; Wallner et al.
1995; Bond et al. 1995). The most interesting observations were that microbes belonging to the c subgroup of
the proteobacteria seemed to be less abundant in activated sludge than was observed by culture-dependent
techniques, and that gram-positive bacteria with a high
G+C DNA content seemed to occur more frequently
than previously suggested. It was also observed (Wagner
et al. 1993, 1994a) that b-subclass proteobacteria were
found more commonly in hybridization studies in situ
than in culture-dependent control experiments using
nutrient-rich media (Luria-Bertani medium or trypticase/soy/agar). Bacteria belonging to this group were,
however, frequently observed in earlier studies using
more dilute media (Dias and Bhat 1964; Benedict and
Carlson 1971; Seiler and Blaim 1982; Blaim et al. 1984)
and therefore the signicance of this observation is
unclear. Thus, the relationship between the results
74
75
approximately 100 ng puried template DNA. The primers used in
the amplications have been reported by Lane et al. (1985). Universal primer C, 5ACGGGCGGTGTGTRC (where R = A/G),
and a primer complementary to universal primer A, 50 CAGCMGCCGCGGTAATWC (where M = A/C; W = A/T), were
used for direct amplication of partial rRNA genes from sample
1a. The amplication product covered the area 5191406 of the 16S
rRNA (primers included) according to the E. coli numbering system and its length was approximately 900 bp. The primers used for
the cultivated isolates were universal primer C and a primer
complementary to primer B, 50 AAACTYAAAKGAATTGACCC
(where Y = C/T; K = G/T). The length of the amplication
product was approximately 500 bp covering the area 9071406 of
the 16S rRNA (according to the E. coli numbering system). The
reaction mixtures were covered with mineral oil and placed in a
thermocycler. After an initial denaturation at 95 C for 5 min, 25
cycles were carried out: 95 C for 1 min, 48 C for 1 min and 72 C
for 30 s. The amplication products were puried by phenol and
chloroform/isoamyl alcohol (24:1) extractions following electrophoretic separation and excision from agarose gels.
To prepare the 16S rRNA gene library from sample 1a, the
puried PCR products were treated with T4 polynucleotide kinase
(Promega) and ligated into SmaI-digested pUC18 cloning vector
using T4 DNA ligase (Promega) according to the manufacturer's
instructions. The ligation products were transformed into
competent E. coli DH5a cells and plated on Luria-Bertani agar
plates (Sambrook et al. 1989) containing 150 lg ampicillin ml)1
and 50 lg 5-bromo-4-chloro-3-indolyl b-D-galactoside ml)1. Plasmids were extracted according to the method of Birnboim and
Doly (1979) and inserts were detected from SmaI digests by agarose
gel electrophoresis. Both strands of the plasmid inserts were sequenced manually using the Sanger dideoxy-DNA method (Sambrook et al. 1989) with Sequenase version 2.0 T7 DNA polymerase
(USB). Primer C and the primer complementary to primer B were
used in sequencing.
The puried PCR products of the cultivated isolates were sequenced manually using a CircumVent thermal cycle sequencing kit
(New England Biolabs) with approximately 3050 ng template
DNA. Both strands of the amplication products obtained were
sequenced using primer C and the primer complementary to primer
B. Reaction conditions were chosen according to the manufacturer's instructions and 20 cycles of amplication were carried out:
95 C for 20 s, 55 C for 20 s and 72 C for 20 s. The amplication
products were analyzed in 7% (w/v) acrylamide gels according to
Sambrook et al. (1989).
Data analysis
The similarity values presented in Table 2 were obtained from
GenBank comparisons. Other similarity values appearing in the
text were calculated using PC/GENE computer software (Bairoch
1992).
Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper will appear in
the GenBank nucleotide sequence database with the following accession numbers: AI047, U45667; AI049, U45687; AI051, U45691;
AI085, U45690; AI111, U46932; AI138, U45666; AI171, U45668;
AI208, U45669; AI235, U45670; AI269, U45671; AI271, U45672;
AI289, U45673; AI301, U45674; AI303, U45675; AI312, U45676;
AI313, U45677; AI346, U45678; AI353, U45679; AI356, U45680;
AI359, U45681; AI361, U45682; AI372, U45683; AI383, U45684;
AI391, U45685; AI423, U45686; AI491, U45688; AI563, U45689;
CL01, U45698; CL02, U45699; CL03, U45700; CL04, U45701;
CL05, U45692; CL06, U45693; CL07, U45694; CL08, U45695;
CL09, U45696 and CL10, U45697. For the accession numbers of
previously published GenBank sequences see Table 2.
Results
Success and reproducibility of the SDS-PAGE analysis
A total of 150 isolates were originally picked from each
subsample. In subsamples 1a, 1b, 2a and 2b the analysis
of whole-cell protein patterns separated by SDS-PAGE
was successful for 138, 139, 122 and 117 isolates respectively. The number of isolates analyzed was decreased for two reasons: (i) several isolates were lost
during the single-colony isolation procedure and (ii) no
bands were observed in some of the protein patterns,
possibly because of inecient cell disruption during
sample preparation.
The grouping of the 516 protein patterns obtained
with the UPGMA algorithm separated 53 protein
groups altogether (Table 1). Subsamples 1a and 1b as
well as subsamples 2a and 2b were collected independently to control the reproducibility of our sampling
procedure and protein pattern analysis. Comparison of
subsamples 1a and 1b showed that 6 out of the 27 protein groups were common to both subsamples. Although
this number was relatively low, a more detailed analysis
of the data indicated that all protein groups with more
than 3 isolates contained isolates from both subsamples
(Table 1). A similar analysis with subsamples 2a and 2b
showed that 22 out of 43 protein groups observed were
common to both subsamples. Again all large protein
groups (with more than 4 isolates) were observed in both
subsamples (Table 1). These analyses conrmed that our
sampling procedure was adequate for culturable organisms in terms of the reproducibility of the results, and
therefore subsamples 1a and 1b, and 2a and 2b were
combined to form samples 1 and 2 respectively.
Comparison of samples 1 and 2
The grouping of isolates from sample 1 showed that
protein groups 1 and 2 contained 204 and 29 isolates
respectively. These gures represent 73% and 10% of
all isolates. Thus, the remaining 25 protein groups
contained only about 17% of isolates in sample 1
(Table 1).
The grouping of sample 2 isolates revealed more
equal distribution (Table 1), the largest protein groups
being 3 (containing 16% of isolates), 1 (9%), 2 (9%), 4
(9%) and 5 (8%). In addition, protein groups 9 (5%), 6
(4%), 8 (4%) and 7 (4%) were composed of at least 9
isolates in sample 2 (Table 1).
The comparison of samples 1 and 2 revealed that 15
protein groups (of 53) were shared by the two samples
(Table 1). Almost all groups with 10 or more isolates
contained isolates originating from both samples, the
only exceptions being protein groups 28 and 32, which
were composed of isolates from the second sample only
(Table 1).
76
Table 1 Distribution of isolates into dierent protein groups
Protein
group
Samples
1a
1b
2a
2b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
112
13
0
0
0
2
0
0
0
0
1
1
1
2
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
92
16
2
1
0
0
3
1
0
2
3
5
1
2
1
0
0
0
1
3
0
0
0
2
1
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
17
20
12
8
7
6
6
5
4
2
2
4
1
2
4
0
2
1
0
1
0
0
0
1
0
1
1
1
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
1
14
5
18
10
11
3
3
4
6
4
3
1
1
1
3
2
4
1
1
0
2
1
0
0
0
1
1
1
1
2
2
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
226
51
40
23
19
12
12
11
11
10
9
9
7
6
6
6
4
3
3
3
3
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
138
139
122
117
516
77
Table 2 Comparison of partial 16S rRNA gene sequences to those
of the GenBank database. PCR polymerase chain reaction; isolates
or clones with identical sequences are indicated by /. The percenProtein
group
Isolate or
PCR clone
Length of
sequences
(bp)
Taxonomic
aliation
Percentage
sequence
similarity
AI049/AI051/AI313
AI359
384/500/411
377
98.4
95.8
AI085/AI138/AI171
343/366/351
AI271
Methanotrophic sp. strain ``B-3060'' (L20845)
96.2
93.4
AI208/AI235
365/366
99.5
AI269/AI312
380/390
98.7
AI346/AI372
364/346
98.9
98.4
AI423/AI563
379/346
100.0
AI271
364
AI353
371
96.2
91.3
90.6
AI301/AI303
358/398
CL08
Leptothrix discophora ATCC 51168 (L33974)
99.5
96.0
AI383/AI391
366/365
98.9
11
AI111
349
98.0
97.7
12
AI289
371
100.0
14
AI047
367
100.0
15
AI361
361
98.9
16
AI356
369
96.7
18
AI491
364
CL01
CL02
CL03
361
368
373
98.9
97.8
97.2
95.7
95.7
CL04
CL05
CL06
355
392
402
CL07
CL08
408
366
CL09
CL10
383
423
b
High G+C a
b
a
b
b
b
b
a
b
96.9
99.0
99.3
96.5
98.3
99.5
95.9
87.8
99.3
97.2
78
Discussion
In this study two bacterial culture collections were obtained from activated sludge. The culturable population
was dierent at dierent sampling times, although no
apparent changes in the plant operation were observed
during the study. The rst sample was dominated by one
culturable (in TGY medium) protein group. In contrast,
in the second sample the distribution of isolates among
protein groups was more equal and no major protein
groups were observed. The latter case has also been
observed in samples analyzed previously from another
wastewater treatment plant (Hantula et al. 1991a). The
question emerging is whether these observations reect
true dierences in diversity, or result from biases caused
by the culture-dependent techniques used.
According to partial 16S rRNA gene sequence comparisons, all the cultivated isolates were related to recognized phyla of the domain bacteria, that is, the a, b
and c subclasses of the proteobacteria. This supports the
results of in situ hybridization studies, which have revealed that proteobacteria account for about 80% of all
79
References
Allen LA (1944) The bacteriology of activated sludge. J Hyg
(Camb) 43: 424431
Amann R, Springer N, Ludwig W, Gortz H-D, Schleifer K-H
(1991) Identication in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351: 161163
Amann R, Ludwig W, Schleifer K-H (1995) Phylogenetic identication and in situ detection of individual microbial cells without
cultivation. Microbiol Rev 59: 143169
American Public Health Association (1985) standard methods for
the examination of water and wastewater, 16th ed. American
Public Health Association, Washington, D.C.
Bairoch A (1992) PC/GENE: the nucleic acid and protein sequence
analysis software system. University of Geneva, Switzerland,
(TM) IntelliGenetics Inc. release 6.70
Benedict RG, Carlson DA (1971) Aerobic heterotrophic bacteria in
activated sludge. Water Res 5: 10231030
Birnboim HC, Doly J (1979) A rapid alcaline extraction procedure
for screening recombinant plasmid DNA. Nucleic Acids Res 7:
15131523
Blaim H, Seiler H, Baumgarten J (1984) Microbial population in an
activated sludge treatment plant of a chemical combine. Z
Wasser Abwasser Forsch 17: 3741
Bond PL, Hugenholz P, Keller J, Blackall L (1995) Bacterial
community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl Environ Microbiol 61: 19101916
Burks C, Cinkosky MJ, Fisher WM, Gilna P, Hayden JE-D, Keen
GM, Kelly M, Kristoerson D, Lawrence J (1992) GenBank.
Nucleic Acids Res 20: 20652069
Buttereld CT (1935) Studies of sewage purication II. A zoogloeaforming bacterium isolated from activated sludge. Public
Health Rep 50: 12671274
DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains:
ribosomal-RNA-based probes for the identication of single
cells. Science 243: 13601363
Dias FF, Bhat JV (1964) Microbial ecology of activated sludge.
Appl Microbiol 12: 412417
Fuhrman JA, McCallum K, Davis AA (1992) Novel major archaebacterial group from marine plankton. Nature 356: 148149
Giovannoni SJ, Britschi TB, Moyer CL, Field KG (1990) Genetic
diversity in Sargasso Sea bacterioplancton. Nature 345: 6063
Gosink JJ, Staley JT (1995) Biodiversity of gas vacuolate bacteria
from Antarctic sea ice and water. Appl Environ Microbiol 61:
34863489
Hantula J, Kurki A, Vuoriranta P, Bamford DH (1991a) Rapid
classication of bacterial strains by SDS-polyacrylamide gel
electrophoresis: population dynamics of the dominant dispersed
phase bacteria of activated sludge. Appl Microbiol Biotechnol
34: 551555
Hantula J, Kurki A, Vuoriranta P, Bamford DH (1991b) Ecology
of bacteriophages infecting activated sludge bacteria. Appl
Environ Microbiol 57: 21472151
Koivula TT, Hantula J (1997) Diversity within bacterial isolates
hybridizing with Comamonas probe ppT. J Basic Microbiol 37:
129137
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR
(1985) Rapid determination of 16S ribosomal RNA sequences
for phylogenetic analyses. Proc Natl Acad Sci USA 82: 6955
6959
Manz W, Wagner M, Amann R, Schleifer K (1994) In situ characterization of the microbial consortia active in two wastewater
treatment plants. Water Res 28: 17151723
Olkkonen VM, Bamford DH (1989) Quantitation of the adsorption
and penetration stages of bacteriophage /6 penetration. Virology 171: 229238
Olsen GJ, Woese CR, Overbeek R (1994) The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol 176: 16
Palleroni NJ (1994) Some reections on bacterial diversity. ASM
News 60: 537540
Prakasam TBS, Dondero NC (1967) Aerobic heterotrophic bacterial populations of sewage and activated sludge. Appl Microbiol
15: 461467
Russel R, Bartow E (1916) Bacteriological study of sewage purication by aeration. Univ Ill Bull, State Water Surv Ser 13: 348
358
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY
Seiler H, Blaim H (1982) Population shifts in activated sludge
from sewage treatment plants of the chemical industry: a numerical cluster analysis. Eur J Appl Microbiol Biotechnol 14:
97104
Sneath PHA, Sokal RR (1973) Numerical taxonomy: the principles
and practice of numerical classication. Freeman, San Francisco
Tsai Y-L, Olson BH (1991) Rapid method for direct extraction of
DNA from soil and sediments. Appl Environ Microbiol 57:
10701074
Wagner M, Amann R, Lemmer H, Schleifer K-H (1993) Probing
activated sludge with oligonucleotides specic for proteobacteria: inadequacy of culture-dependent methods for describing
microbial community structure. Appl Environ Microbiol 59:
15201525
Wagner M, Erhart R, Manz W, Amann R, Lemmer H, Wedi D,
Schleifer K-H (1994a) Development of an rRNA-targeted
oligonucleotide probe specic for the genus Acinetobacter and
its application for in situ monitoring in activated sludge. Appl
Environ Microbiol 60: 792800
Wagner M, Assmus B, Hartmann A, Hutzler P, Amann R (1994b)
In situ analysis of microbial consortia in activated sludge using
uorecently labelled, rRNA-targeted oligonucleotide probes
and confocal scanning laser microscopy. J Microsc 176: 181
187
Wallner G, Erhart R, Amann R (1995) Flow cytometric analysis of
activated sludge with rRNA-targeted probes. Appl Environ
Microbiol 61: 18591866
Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O,
Krichevsky MI, Moore LH, Moore WEC, Murray RGE,
Stackebrandt E, Starr MP, Truper HG (1987) Report of the ad
hoc committee on reconciliation of approaches to bacterial
systematics. Int J Syst Bacteriol 37: 463464
Woese CR (1987) Bacterial evolution. Microbiol Rev 51: 221271